Multiplex Nucleic Acid Detection

ABSTRACT

The present invention relates to a novel multiplex detection of nucleic acid targets in a sample. This is accomplished by using novel padlock probes which contain a unique cleavage site for linearization and a first member of a binding pair for isolation of the padlock probe from the sample. Three different designs of the padlock probes are described and examples are shown for ligation, amplification and qualitative and quantitative detection. The combination of these features gives a fast, accurate and specific multiplex detection assay

The invention relates to multiplex detection of nucleic acid targets,more specifically detection on micro-arrays with tagged probes, evenmore specifically where such probes are padlock probes.

The accurate identification and detection of (pathogenic)micro-organisms or other targets of interest has become increasinglyimportant in clinical diagnostics and pest management strategies.Traditionally, the predominant techniques used to identify pathogenshave relied upon culture-based morphological approaches. These classicaltools have been complemented in recent years by variousculture-independent molecular characterizations, especially thoseinvolving PCR amplification of pathogen-specific nucleic acid targets(Atkins, S. D. and Clark, I. M. (2004) Fungal molecular diagnostics: amini review. J. Appl. Genet., 45, 3-15; Lopez, M. M., Bertolini, E.,Olmos, A., Caruso, P., Gorris, M. T., Llop, P., Penyalver, R. andCambra, M. (2003) Innovative tools for detection of plant pathogenicviruses and bacteria. Int. Microbiol., 6, 233-243.). Although theseapproaches are generally effective, they target only a single pathogenper assay, making comprehensive screening of samples laborious andtime-consuming.

To increase efficiency, it is desirable to develop multiplex assays,which can detect several targets simultaneously. Microarrays may enablehighly parallel detection of diverse organisms (Bodrossy, L. andSessitsch, A. (2004) Oligonucleotide microarrays in microbialdiagnostics. Curr. Opin. Microbiol., 7, 245-254.). Typically, multiplexstrategies involve either amplification with generic primers that targeta genomic region containing species-specific information or multipleprimer sets. Although such strategies are a marked improvement overtraditional PCR-based assays, there are still serious limitations.Targeting a conserved genome region limits the analysis to ataxonomically defined group of pathogens, while combining several primersets may present a significant technical challenge. Recently, universalamplification coupled with microarray analysis was suggested as anunbiased approach to pathogen detection (Vora, G. J., Meador, C. E.,Stenger, D. A. and Andreadis, J. D. (2004) Nucleic acid amplificationstrategies for DNA microarray-based pathogen detection. Appl. Environ.Microbiol., 70, 3047-3054.). Although it overcomes the above-mentionedproblems, the sensitivity at present is not sufficient for diagnosticpurposes.

Padlock probes (PLP) are circularising probes which can offer a means ofcombining specific molecular recognition and universal amplification (orspecific amplification and general recognition), thereby increasingsensitivity and multiplexing capabilities without limiting the range ofpotential target organisms. PLPs are long oligonucleotides ofapproximately 100 bases, containing target complementary regions at boththeir 5′ and 3′ ends (FIG. 1). These regions recognise adjacentsequences on the target DNA (Nilsson, M., Malmgren, H., Samiotaki, M.,Kwiatkowski, M., Chowdhary, B. P. and Landegren, U. (1994) Padlockprobes: circularizing oligonucleotides for localized DNA detection.Science, 265, 2085-2088.), and between these segments lie universalprimer sites and a unique sequence identifier, the so-called ZipCode.Upon hybridisation, the ends of the probes get into adjacent position,and can be joined by enzymatic ligation converting the probe into acircular molecule that is threaded on the target strand. This ligation,and the resulting circular molecule can only take place when both endsegments recognise their target sequences correctly. Non-circularisedprobes are removed by exonuclease treatment, while the circularised onesmay be amplified with universal primers. This mechanism ensures reactionspecificity, even in a complex nucleotide extract with a large number ofpadlock probes. Subsequently, the target-specific products are detectedby a universal cZipCode microarray (Shoemaker, D. D., Lashkari, D. A.,Morris, D., Mittmann, M. and Davis, R. W. (1996) Quantitative phenotypicanalysis of yeast deletion mutants using a highly parallel molecularbar-coding strategy. Nat. Genet., 14, 450-456.). PLPs have been shown tohave good specificity and very high multiplexing capabilities ingenotyping assays (Hardenbol, P., Baner, J., Jain, M., Nilsson, M.,Namsaraev, E. A., Karlin-Neumann, G. A., Fakhrai-Rad, H., Ronaghi, M.,Willis, T. D., Landegren, U. and Davis, R. W. (2003) Multiplexedgenotyping with sequence-tagged molecular inversion probes. Nat.Biotechnol., 21, 673-678.).

The inventors now have found an efficient and reliable multiplexamplification and detection system, which makes use of improved,specifically designed padlock probes.

The present invention gives a solution for one of the problemsassociated with the use of padlock probes (PLPs), especially inmultiplex assays, which is background amplification of non ligated PLP'sduring the consecutive PCR step.

To solve this, the invention comprises a padlock oligonucleotide probecomprising (from 5′ to 3′):

a) a target specific nucleotide sequence 1 (T1);

b) a generic reverse primer binding site;

c) a nucleotide sequence acting as or bearing a first member of abinding pair;

d) a unique cleavable sequence;

e) a generic forward primer binding site;

f) a ZIP-code sequence; and

g) a target specific nucleotide sequence 2 (T2),

wherein the T1 and T2 sequences are designed to be complementary toadjacent nucleotide stretches on the same target in such a way thatafter hybridization (and ligation of the outer ends) the padlock probeforms a circular molecule. The functionalities of item c) and d) can becombined, i.e. the unique cleavable sequence can serve as a nucleotidesequence acting as a first member of a binding pair. Alternatively, thefirst member of a binding pair is a desthiobiotin moiety, and thenucleotide bearing said desthiobiotin moiety is preferably coupled tothe poly-uracil sequence through a thymidine linker. The uniquecleavable sequence is preferably a mono-nucleotide sequence, such as apoly-uracil sequence. The generic forward primer binding site cancomprise a T7 RNA polymerase recognition site. Preferred is anembodiment, wherein the ZIP code sequence is the complementary strand ofa nucleotide sequence on an array.

Another aspect of the present invention is a padlock nucleotide probecomprising from 5′ to 3′

a) a target specific nucleotide sequence 1 (T1);

b) a unique reverse primer binding site;

c) a nucleotide sequence acting as or bearing a first member of abinding pair;

d) a unique cleavable sequence;

e) a unique forward primer binding site; and

f) a target specific nucleotide sequence 2 (T2),

wherein the T1 and T2 sequences are designed to be complementary toadjacent nucleotide stretches on the same target in such a way thatafter hybridization (and ligation of the outer ends) the padlock probeforms a circular molecule. The functionalities of item c) and d) can becombined, i.e. the unique cleavable sequence can serve as a nucleotidesequence acting as a first member of a binding pair. Alternatively, thefirst member of a binding pair is a desthiobiotin moiety. The uniquecleavable sequence is preferably a mono-nucleotide sequence, such as apoly-uracil sequence. Optionally, the padlock nucleotide probe of thisembodiment contains a universal ZIP code sequence, preferably betweenthe unique forward primer binding site and the T2 sequence.

The invention also provides a method for the detection of a targetnucleotide sequence comprising:

a. adding to a sample which contains said target, one or more padlocknucleotide probes of the invention, which have target specific sequencescapable of hybridisation to said target;

b. allowing annealing of the padlock nucleotide probe to said target;

c. circularisation of padlock probe by ligation;

d. capturing the padlock nucleotide probes by bringing them into contactwith a solid support coated with a second member of a binding pair

e. linearising the padlock nucleotide probe by cleaving the uniquecleavable sequence;

f. washing of the beads to remove any unbound oligonucleotides

g. elution of the PLP probe from the solid support

h detection of the ZIP-code sequence.

In said method the detection of the ZIP-code preferably comprises thesteps of:

i. amplification of the padlock nucleotide probe using the genericprimers;

j. labelling the amplified padlock nucleotide probe;

k. testing for presence of the ZIP-code by hybridising said padlocknucleotide probe with at least one sequence which is capable ofhybridisation with said ZIP-code sequence, wherein said hybridisationpreferably takes place at a solid support, such as a (micro-) array.

Alternatively in said method the detection of the ZIP-code can beperformed also directly:

-   -   i. testing for presence of the ZIP-code by hybridising said        padlock nucleotide probe with at least one sequence which is        capable of hybridisation with said ZIP-code sequence, wherein        said hybridisation preferably takes place at a solid support,        such as a (micro-) array or gold beads.    -   j. labelling of the hybridized padlock nucleotide probe on the        solid support with a fluorescent probe directed against the        first member of a binding pair of the padlock or with        fluorescently labelled Biobarcode labelled gold beads.

These methods preferably comprise an NaOH denaturation step beforecapturing of the padlock nucleotide probes. The second member of abinding pair will bind the first member of a binding pair which isavailable on the PLP. If said first member is desthiobiotin, said secondmember is streptavidin. In the case of direct detection on the array,the elution of step g. is performed with biotin or with heat. If theunique cleavable sequence is a poly-uracil sequence, cleavage canpreferably be effected by treatment with uracil-N-glycosidase andendonuclease IV.

In the case of BCA detection, the linearized PLP stays bound to thestreptavidin and goldbeads bearing both ZIP codes complementary to thenucleotide sequence in the padlock and fluorescent barcode nucleotides,will be hybridized.

Another aspect of the invention is a method for the detection of atarget nucleotide sequence comprising:

a. adding to a sample which contains said target, one or more padlocknucleotide probes, which have target specific sequences capable ofhybridisation to said target;

b. allowing annealing of the padlock nucleotide probe to said target;

c. circularisation of padlock probe by ligation;

d. capturing the padlock nucleotide probe using a solid support coatedwith a second member of a binding pair;

e. linearising the padlock nucleotide probe by cleaving the uniquecleavable sequence;

f. eluting the padlock nucleotide probe from the solid support,

g. amplifying the eluted padlock nucleotide probe using the uniqueprimers;

h. monitoring and detecting amplification.

In said method, the amplification and monitoring of the amplification ispreferably performed on an Open Array™ system (BioTrove). The method canoptionally comprise an NaOH denaturation step before capturing of thepadlock nucleotide probes.

The second member of a binding pair will bind the first member of abinding pair which is available on the PLP. If said first member isdesthiobiotin, said second member is streptavidin In that case, theelution of step f. is performed with biotin.

If the unique cleavable sequence is a poly-uracil sequence, cleavage canpreferably be effected by treatment with uracil-N-glycosidase andendonuclease IV.

A further aspect of the invention is the use of padlock nucleotideprobes according to the invention for the multiplex detection ofnucleotide sequences. A next aspect of the invention is a test kitcomprising multiple padlock probes according to the invention, whereineach padlock probe is designed to recognise a unique target.

DESCRIPTION OF THE FIGURES

FIG. 1. Scheme of conventional PLP ligation and real-time PCR toquantify single mismatch discrimination. (A) PLPs containtarget-complementary sequences at the 5′ and 3′ ends (T1, T2), flankingthe universal primer sites (P1, P2) and the unique identifier ZipCodesequence (Zip). (B) T1 and T2 bind to adjacent sequences on the target,and in the case of a perfect match, the probe may be circularised by aligase (a). Mismatch-containing molecules are expected to bediscriminated, and no ligation should occur (b). (C) Unreacted probesare removed by exonuclease treatment. (D) Circularised probes areamplified using two universal primers and amplification is monitored inreal-time using a TaqMan probe, which detects the ZIP-code. (E) Ligationyields with different target oligonucleotides can be accuratelyquantified based on the threshold cycle (Ct) values of amplification.

FIG. 2. Amplification curves of a representative experiment to optimizePLP design for mismatch discrimination. The respective ligation targetsare indicated for each sample (Table 2A). In the insert the calibrationcurve of the real-time PCR is shown, which was used to determineamplification efficiency (E=0.81).

FIG. 3. Sensitivity and discriminatory range of diagnostic PLPs wereassessed by using synthetic complementary oligonucleotides and genomicDNAs.

(A) PLP P-inf was ligated on serial dilutions of oligonucleotidesrepresenting closely related, non-target and target sequences,respectively. Reactions were scored as either negative or positive, asshown in the table below (‘nt’ stands for ‘not tested’). Detectionthreshold is indicated by an asterisk and the magnitude ofdiscriminatory range is shown. (B) Detection of dilution series of P.nicotianae and P. cactorum genomic DNAs using the corresponding PLPs.Template DNAs are indicated above the picture, while the used PLPs areshown below. (C) Ligation of PLP P-cac on genomic DNA of P. nicotianaedoes not result in a positive signal even in the presence of very highamount of DNA. Amount of template DNAs are shown above the picture.

FIG. 4. Layout of multi-chamber universal tag array. Deposition schemeand sequences of cZipCode probes.

FIG. 5: Schematic approach of the traditional PLP technology fordetection of pathogens (for details see text).

FIG. 6. Detection of genomic DNAs corresponding to individual (a-g) andcomplex pathogen samples (h-l) on a universal microarray. The analyzedtargets were: (a) P. cactorum; 1 ng (b) P. nicotianae, 1 ng; (c) P.sojae, 1 ng; (d) R. solani AG 4-2, 1 ng; (e) M. hapla, 1 ng; (f) F.oxysporum, 1 ng; (g) M. roridum, 1 ng; (h) Pyt. ultimum, 500 pg; M.hapla, 500 pg and P. nicotianae, 500 pg; (i) P. infestans, 500 pg; R.solani AG 4-2, 500 pg and M. roridum, 500 pg; (j) P. cactorum, 500 pg;R. solani AG 4-1, 500 pg and V. dahliae, 500 pg. (k) F. oxysporum, 0.5pg and M. roridum, 500 pg; (1) F. oxysporum, 500 pg and M. roridum 5 pg;

FIG. 7. Three preferred probes of the invention, the standard Padlockprobe, the PRI-lock probe and the LUNA-probe.

FIG. 8 Schematic approach of PLP technology with the newly designedstandard PLPs for detection of pathogens (for details see text)

FIG. 9. Schematic overview PRI-lock probe based amplification combinedwith generic TaqMan detection (for details see text)

FIG. 10. Schematic overview of the PRI-lock probe based multiplexdetection in combination with the Open Array™ system (BioTrove).

FIG. 11. Sequences of the designed PRI-lock probes and of the universalTaqMan probe. Different parts of the PRI-locks are indicated bydifferent lettertypes. Bold: target complementary sites. Italic: reverseprimer binding site. Underlined: forward primer binding site. Gray box:the universal TaqMan probe region. The deoxy-uracil cleavage site, thelinker and the desthiobiotin moiety are indicated in open boxes. LNA(locked nucleic acid) nucleotides in the TaqMan probe sequence are shownin capitals

FIG. 12. Amplification plots of real-time PCR performed on ligatedPRI-lock probes.

The data points for PRI_M.ror, PRI_Phyt and PRI_P.inf are shown in ◯, ⋄and Δ, respectively.

FIG. 13. Calibration curves for target quantification using PRI-lockprobe ligation and subsequent real-time PCR. The Ct values were plottedin function of log2 (input DNA concentration in the ligation, expressedas fg/μL). The data points and equations for PRI_M.ror, PRI_Phyt andPRI_P.inf are shown in ▴, ♦ and ▪ respectively. Data points which werenot in the linear range of quantification were omitted from calibrationcurve equations (open symbols).

FIG. 14. Scheme for array based and solution based multiplex detectionwith LUNA probes.

FIG. 15. Principle of NASBA reaction in combination with MolecularBeacon (AmpliDet RNA).

FIG. 16. Examples of two LUNA probes for the detection of Verticilliumdahliae and Phytopthora cactorum., the targets in NASBA the produced RNAamplicons and two specific molecular beacons for the produced RNA's

Different parts of the LUNA probes are indicated by differentlettertypes. Bold: target complementary sites. Small letters: reverseprimer binding site. Under lined, Italic and in capital: T7 recognitionsite. Small letters and underlined: forward primer binding site. Graybox: the specific hybridization site. The deoxy-uracil cleavage site,the linker and the desthiobiotin moiety are indicated in open boxes.

FIG. 17. Amplification plots of real-time NASBA and Molecular Beacondetection performed with two ligated LUNA probes showing specificity andsensitivity.

FIG. 18. Amplification plots of real-time NASBA and Molecular Beacondetection performed with two ligated LUNA probes showing sensitivity anddynamic range.

FIG. 19. Application scheme for the LUNA technology. After LUNA probehybridization, ligation, exonuclease and glycosidase treatment, NASBA isperformed. Visualization of the produced NASBA RNA amplicons can beperformed on array, Luminex beads or with Molecular Beacons.

FIG. 20. N-glycosidase—Endo IV cutting of the PRI lock probes withdifferent length of spacer.

FIG. 21 Schematical view of complex DNA samples which are genericallyamplified by pre-amplification with Phi29, tandem Klenow or ribosomalPCR followed by a ligation detection reaction (LDR) using padlockprobes.

FIG. 22 Detection of ribosomal PCR preamplified genomic DNAs with theligation detection reaction. The analyzed targets were: A. tumefaciens,M. hapla and V. dahliae

FIG. 23 DNA_BCA assay (A) Nano particle and magnetic microparticle probepreparation. (B) Nano particle-based PCR less DNA amplification scheme(according to Jwa-Min Nam, Savka I. Stoeva and Chad A. Mirkin.Bio-Bar-Code-Based DNA Detection with PCR-like Sensitivity. JACS, 126,5932-5933 (2004)).

FIG. 24 Principle of Bio-barcode based signal amplification of a ligatedstandard PLP.

FIG. 25 Principle of Bio-barcode based multiplex signal amplification,after capturing of different ligated standard PLPs.

DEFINITIONS

The term “hybrid” refers to a double-stranded nucleic acid molecule, orduplex, formed by hydrogen bonding between complementary nucleotides.The terms “hybridise” or “anneal” refer to the process by which singlestrands of nucleic acid sequences form double-helical segments throughhydrogen bonding between complementary nucleotides.

The term “ligation” refers to the process of enzymatically joining twoor more nucleotide sequence together by coupling the 5′ P moiety of onenucleotide to the 3′ OH moiety of a second nucleotide, thereby leavingthe polynucleotide backbone intact, which thus will result in aconcatenated, normal nucleotide sequence. The enzyme used for ligationis a ligase.

By “amplification” is meant the construction of multiple copies of anucleic acid sequence or multiple copies complementary to the nucleicacid sequence using at least one of the nucleic acid sequences as atemplate. Methods of the invention can in principle be performed byusing any nucleic acid amplification method, such as the PolymeraseChain Reaction (PCR; Mullis 1987, U.S. Pat. Nos. 4,683,195, 4,683,202,and 4,800,159) or by using amplification reactions such as Ligase ChainReaction (LCR; Barmy 1991, Proc. Natl. Acad. Sci. USA 88:189-193; EPAppl. No., 320,308), Self-Sustained Sequence Replication (3SR; Guatelliet al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), StrandDisplacement Amplification (SDA; U.S. Pat. Nos. 5,270,184, and5,455,166), Transcriptional Amplification System (TAS; Kwoh et al.,Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal., 1988, Bio/Technology 6:1197), Rolling Circle Amplification (RCA;U.S. Pat. No. 5,871,921), Nucleic Acid Sequence Based Amplification(NASBA), Cleavage Fragment Length Polymorphism (U.S. Pat. No.5,719,028), Isothermal and Chimeric Primer-initiated Amplification ofNucleic Acid (ICAN), Ramification-extension Amplification Method (RAM;U.S. Pat. Nos. 5,719,028 and 5,942,391) or other suitable methods foramplification of DNA. The product of amplification is termed anamplicon. Amplification as used in the present invention also comprisesBioBarCode amplification (BCA) as described by Jwa-Min Nam, Savka I.Stoeva and Chad A. Mirkin. Bio-Bar-Code-Based DNA Detection withPCR-like Sensitivity. JACS, 126, 5932-5933 (2004). Although this is notan amplification in the sense that multiple copies of the original aremade, it constitutes an amplification of the signal.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target (the “primer bindingsite”) allowing a polymerase to attach thereby serving as a point ofinitiation of DNA or RNA synthesis when placed under conditions in whichsynthesis of primer extension product which is complementary to anucleic acid strand is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxy ribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and source of primer. Normally, primerscome in sets including one forward and one reverse primer as commonlyused in the art of DNA amplification such as in PCR amplification. Thusalso the “primer binding sites” on the target DNA are present in a setof one for the forward and one for the reverse primer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention gives a solution for one of the problemsassociated with the use of padlock probes (PLPs), especially inmultiplex assays, which is background amplification of non ligated PLP'sduring the consecutive PCR step. Padlock probes also have some tendencyto linear dimer formation as a result of cross reactive ligation, thecorresponding ligation products can easily be distinguished fromcircularized probes by exonucleolytic degradation. The exonucleasetreatment reduces the number of such linear monomeric and dimericmolecules by almost three orders of magnitude with negligible effects oncircularized probes.

Removal of unreacted probes further reduces ligation independentamplification events that may otherwise occur through accidental primersor templating of polymerization by the large number of linear probes.This can be effected by linearizing the exonuclease treated probes bycleavage at a unique site, which was introduced in the probe. It isparamount that the cleavage site is unique to prevent multipleoccurrences in the probe or in the target nucleotides. Thus, it would bepossible to use restriction sites which are recognized and cleaved by arestriction enzyme, but in that case the site should be basicallynon-existing in the sample nucleotide material and only occur once ineach padlock probe. A few examples of such a sequence would beCTAAGNNNNNCTTAG (wherein N denotes any nucleotide), which is cleavableby the enzyme C EcoO109I; the sequence TGGCGACGAAAACCGCTTGGAAAGTGGCTG,which is cleavable by the enzyme F-TflI; ACCTACCATTAACGGAGTCAAAGGCCATTG,which is cleavable by the enzyme F-TflII,TAGGTACTGGACTTAAAATTCAGGTTTTGT, which is cleavable by the enzymeF-TflIII; CAAAACGTCGTAAGTTCCGGCGCG which is cleavable by the enzymeH-DreI; and GAGTAAGAGCCCGTAGTAATGACATGGC, which is cleavable by theenzyme I-BmoI.

However, all these enzymes only cut double-stranded DNA. To make the DNAdouble-stranded a complementary strand to the site that has been builtin the padlock probe should be added, which then anneals to the padlock,forming a short double-stranded sequence. Addition of the restrictionenzyme then will cut the padlock and linearize it.

Alternatively, Hardenbol et al. (supra) described a different solutionfor this problem for use in a single-stranded molecule by using uracildepurination with uracil-N-glycosidase and cleavage of the sugarbackbone with a heat step. However, it was found that this does not leadto an efficient cleavage of the backbone, which thus still leavescontamination during the amplification reaction. Use of EndoIV nucleaseaccording to this invention in stead of the heat step is much moreefficient, thereby reducing background contamination and thus increasingthe sensitivity of the assay.

Thus, in one embodiment, the PLP of the invention preferably contains apoly-uracil site for enabling linearization of the probes. Preferably,the unique cleavage sequence is introduced just to the 5′ side of theunique forward primer binding site, which, after cleavage becomes themost 5′ part of the linearized molecule. In the same way, the uniquereverse primer binding site should be positioned as close as possible 5′of the unique cleavage sequence, in order to leave, upon cleaving, saidreverse primer binding site at the most 3′end of the linearizedmolecule.

In between the two universal primer binding sites then the ligatedtarget recognition sites and the ZIP code will be present, which wouldensure a proper amplification of the parts of the PLP that are used forgiving a specific reaction in the assay.

In case of cleavage of the poly-uracil sequence a treatment withendonuclease IV is performed for efficient cutting the deoxy-ribosephosphate backbone at the ends of the linearized polynucleotide.

Further, linearization of the PLP before amplification ensures that PLPswhich have not been ligated at the target site will not be amplified.They are also cleaved at the unique cleavage site, leaving one shortpiece of DNA with only the unique reverse primer binding site, andanother, a bit longer stretch, bearing the unique forward primer bindingsite. Since those pieces are not joined anymore, an amplification stepusing the universal primer set will not be able to generateamplification products to these incomplete PLPs. Therefore, bylinearizing the PLP, an increase in the detection limit is obtained,since the background (noise) amplification signal will be much lower.

While for specifically recognised restriction sites, the length of thesite is fixed, the length of the poly-uracil sequence is not critical,as long as it gives a good cleavage upon application ofuracil-N-glycosidase and endo IV nuclease. Preferably, the stretch ofuracil nucleotides has at least 2 uracil bases. In principal, there isno upper limit to the length of the poly-uracil sequence, it will inpractice be limited by the technical requirements of the synthesis.

Although also other restriction enzyme sites could be used to linearizethe PLP, a poly-uracil site is preferred because this will normally notbe present in any of the target nucleotides, nor in any of the furtherbuilding blocks of the PLP. Thus, it provides an unique site, withlittle or no chance of disturbing other nucleotides which are present inthe reaction of the assay. Further, use of the poly-uracil enableslinearization of the padlock probe while it is still in thesingle-stranded state and thus, no additional mixing with complementaryoligonucleotides is necessary. Also the used uracil-N-glycosidase andendonuclease IV have no negative effect on the other nucleotides in thereaction

The target molecules, which have to be assayed, can be any form of DNAor RNA, such as genomic DNA, cDNA, mitochondrial DNA, nuclear DNA,messenger RNA, ribosomal RNA and the like. The type of nucleotide isunimportant, but the target should be able of being specificallyrecognised by the corresponding padlock nucleotide probe.

Advantageously, the target nucleotides, which are present in the sampleto be assayed, are randomly cut into smaller fragments of 100-1000basepairs. This can be done using standard methods well known to aperson skilled in the art. Such a random cutting prevents binding of theprobe to very large target molecules, which would (partly) survive theexonuclease treatment.

For an optimal hybridisation reaction to the target nucleotide in thesample it has been found that the melting temperatures (Tm) of the twotarget specific sequences T1 and T2 need to be different. Further, itwas found that asymmetric PLP design, in which a long 5′ arm serves asan anchor sequence and the binding of a short 3′ arm is an equilibriumprocess, could increase mismatch discrimination by almost one order ofmagnitude. Faruqi and colleagues also demonstrated the superiority ofasymmetric PLP design (Faruqi, A. F., Hosono, S., Driscoll, M. D., Dean,F. B., Alsmadi, O., Bandaru, R., Kumar, G., Grimwade, B., Zong, Q., Sun,Z., Du, Y., Kingsmore, S., Knott, T. and Lasken, R. S. (2001)High-throughput genotyping of single nucleotide polymorphisms withrolling circle amplification. BMC Genomics, 2, e4.). Their assayconditions and evaluation method, however, were very different fromthose used in this invention. A further advantage of the asymmetricdesign is that while the 3′ arm may ensure excellent specificity, thebinding of the long 5′ arm is quite stable and might tolerate potentialmismatches caused by polymorphisms within the target group. For asufficient and specific recognition of the target sequence in thesample, the PLP preferably comprises a 5′ arm (T1), which preferably hasa length of about 10 to about 75 nucleotides, more preferably of about20 to about 50 nucleotides and most preferably of about 25 to about 40nucleotides. The shorter 3′ arm (T2) preferably has a length of about 10to about 30 nucleotides, more preferably of about 10 to about 20nucleotides. Examples of such T1 and T2 sequences are given in Table 3A.

The pivotal point of the present invention is that even a betterdetection is achieved when it is possible to isolate the circularizedPLPs from the reaction mixture, which contains not only the ligated fulllength PLPs which have been linearized by cleavage at the uniquecleavage site, but also the unreacted sample nucleotides, and short PLPfragments stemming from non-ligated, cleaved PLPs. Isolation of thecircularised probes is preferably accomplished by incorporation of anucleotide carrying a first member of a binding pair. Isolation of thePLPs can then be achieved by contacting said PLPs with a solid supportcarrying the second member of said binding pair, and thus binding thePLPs. After binding the unreacted nucleotide sequences can be washedaway, whereafter the bound PLPs can be eluted from the solid support.The solid support can be anything which is able to carry the secondmember of the binding pair, such as beads or a column. The material ofthe solid support can be any material which is conventionally used inbiochemical procedures of this kind, such as glass, polystyrene,polyethylene, and the like.

A preferred binding pair is (desthio-)biotin/streptavidin. It has beenfound extremely suitable to provide the PLP with a nucleotide carrying adesthio-biotin moiety. This enables binding to streptavidin coatedmagnobeads (Hirsch J D, Eslamizar L, Filanoski B J, Malekzadeh N,Haugland R P, Beechem J M and Haugland R P. (2002) Easily reversibledesthiobiotin binding to streptavidin, avidin, and other biotin-bindingproteins: uses for protein labeling, detection, and isolation. AnalBiochem. 2002 Sep. 15; 308 (2):343-57) after which the unboundnucleotides can be washed off. The PLPs which are bound to the beads canbe set free again by addition of biotin, which binds more strongly tothe streptavidin coated magnobeads and replaces the PLP. With thissystem it is possible to obtain a rather pure isolate of the PLPs whichhave recognized and hybridised to a target in the original reactionmixture and have been ligated to form circular PLPs. However, any othermembers of a binding pair can be used, such as antigen/antibody, DNA/DNAbinding protein and the like can be used in the present invention, aslong as elution from the solid carrier is possible. Elution can beperformed by addition of a competitive binder (such as biotin in thecase of the desthio-biotin/avidin binding) or by changes in saltconcentration, temperature or ionic strength.

Another preferred embodiment is to use the poly-uracil as a first memberof a binding pair. This can be bound to a poly-A sequence, e.g. providedon a solid support like a column. After washing the column to remove anyunbound nucleotides, the padlock nucleotide probes can be eluted bywashing under increased temperature or with a mildly basic solution (0.1M NaOH). Preferably, the nucleotide bearing the first member of abinding pair, such as the desthio-biotin moiety, is engineered betweenthe unique reverse primer binding site and the unique cleavage site,since there it will not interfere with any subsequent amplificationreaction (it will be at the 3′ end of the reverse primer binding siteand thus remain outside the sequence which is amplified). Further, itneeds a minimal distance from the cleavage site.

We have found that the uracil-N-glycosidase cannot approach the uracilmolecules if the first member of a binding pair (desthio-biotin in theexamples) is bound too close to these nucleotides. We experimentallyfound out that if 12 nucleotides or more are between uracil anddesthiobiotin degradation can take place (FIG. 20). The use of poly T'sis because this does not complicate the PLP design significantly, thisin contrast with a random nucleotide sequence, which can influence thesecondary structure. It is contemplated that the exact sequence of thisspacer is not critical as long as it does not interfere with thesecondary structure and as long as it does not interfere with theamplification reaction and/or the target hybridization reactions. Thus,preferably a stretch of at least 12 identical nucleotides is used.

Contacting the sample with beads or any other support coated with thesecond member of the binding pair, such as streptavidin, will cause thePLPs to bind, while the rest of the sample, inclusive all unboundnucleotides, can be removed, e.g. through washing with buffer solutionor by physically separating the beads. Freeing the PLPs again from thestreptavidin binding can fairly easy be accomplished by a competitionreaction with biotin, which binds stronger to the streptavidin thandesthio-biotin and thus will replace the bound PLPs at the streptavidinebeads. These free PLPs can then be eluted from the solution and thus areobtained in a purified form.

It will be clear to a person skilled in the art that by isolating thePLPs in the above mentioned way, the subsequent amplification will beless prone to false positives.

Preferably, the steps of isolation and linearization of the PLPs, asdescribed above, are combined. This means that the linearization of theprobes, i.e. the cleavage at the poly-uracil site, is performed whilethe PLPs are attached to the streptavidin coated solid support.

After linearization, the PLPs are fit (may serve as template) foramplification. As explained above, the use of padlock probes in generaland specifically in combination with the cleavage at the uracil-siteensures that only the PLPs are amplified which have recognized a targetsequence, been able to hybridise to said target sequence and which havebeen ligated at said target site. Thus by amplifying only those PLPs agenuine representation of the target sequences that were present in theoriginal sample can be obtained.

After amplification the PLPs can be assayed by using any sort of assaywhich is capable of recognising specific polynucleotides. Depending onthe type of PLP the assay can vary (as described below). Preferably,especially in the case of multiplex amplification, the assay isperformed on a (micro-)array. Depending on the choice of the PLP, themethod can be qualitative or quantitative.

In accordance with a quantitative method of the invention, the specificsequence of the PLP (which can for instance be provided by the ZIP-codeor by the target specific sequences T1 or T2) is recognised by aspecific capture molecule (e.g. a sequence which is capable ofhybridisation with said specific sequence), which bears a label. Thestrength of the generated signal is compared to a calibration curveproduced from specific sequences in known amounts. As a result, theamount of target nucleotide sequence in the sample can be calculated.This technique thus uses an external standard.

For labelling, substances such as radioisotopes, fluorescent substances,chemiluminescent substances and substances with fluorophore, and thelike may be used. For instance, the fluorescent substance includes Cy2,FluorX, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, fluorescein isothiocyanate (FITC),Texas Red, Rhodamine, Alexa 532 and the like. Methods to attach thelabels to the nucleotides are generally known in the art.

Alternatively, a quantitative method of the invention relates to aninternal standard. In this method, a known amount of one or more markertarget nucleotide sequences is added to the sample. These will then berecognised by a PLP which is specifically designed for this markertarget nucleotide. The PLP will undergo the same treatment as the PLPswhich have recognised their target nucleotides in the sample. Whenperforming the assay, all PLPs are detected by their specific sequencesand a comparison of the signals generated by the PLP which is directedto the marker target nucleotide with the signals generated by the otherPLPs indicates the relative amount of target present in the sample.Since the concentration of the marker target nucleotide was known, theconcentration of other target nucleotides can be calculated. To decreasethe error margins, several different marker target nucleotides can beadded to the sample in increasing concentrations, to generate a sort ofinternal calibration curve.

In another preferred embodiment of the invention, both the amplificationand the detection of the PLPs is performed in a micro-array. TheOpenArray™ technology (BioTrove, Woburn, Mass., USA) currently allowsparallel amplification and testing of more than 3000 assays on one plate(48 subarrays with each 64 so-called Through-Holes with a volume of 33nL). The primers are pre-loaded into the holes, while the (purified)sample along with the reagents are autoloaded due to surface tension,provided by the hydrophilic surface of the array. Detection of theamplification can take place by simple detection of the presence ofdouble stranded DNA. This can be done by using intercalating dyes, suchas ethidiumbromide (EtBr) and SYBR Green. Alternatively, amplificationproducts can be detected and quantified by using a universal probe, suchas a TaqMan probe. TaqMan probes are probes with fluorescent dyes atopposite ends and can be used during PCR amplification. The probe isdegraded during amplification by 5′-exonuclease activity of theTaq-polymerase used and increase of fluorescence can be measuredreal-time during amplification and in that way quantification of targetis possible (see Livak K J, Flood S J A, Marmaro J, Giusti W and Deetz K(1995). Oligonucleotides with fluorescent dyes at opposite ends providea quenched probe system useful for detecting PCR product and nucleicacid hybridization. PCR Methods and Applications 4: 357-362).

In a preferred embodiment the TaqMan probes for detecting amplificationproducts comprise locked nucleic acids (LNA) nucleotides. Locked nucleicacids (LNAs) are synthetic nucleic acid analogs that bind tocomplementary target molecules (DNA, RNA or LNA) with very highaffinity. At the same time when probes are compared with and without LNAhaving the same Tm, binding affinity is decreased substantially for theLNA type when the hybrids thus formed contain even a single mismatchedbase pair. For this reason LNA existing TaqMan probes show an increasedspecificity (see Koshkin, A. A., Nielsen, P., Meldgaard, M., Rajwanshi,V. K., Singh, S. K. and Wengel, J. (1998) LNA (locked nucleic acid): anRNA mimic forming exceedingly stable LNA:LNA duplexes. J. Am. Chem.Soc., 120, 13252-13253).

In another preferred embodiment of the invention, the detection of theZIP-code can be performed also directly through hybridising the saidpadlock nucleotide to an array or to gold beads bearing ZIP codescomplementary to the nucleotide sequence in the padlock.

In the ligation detection reaction generic pre-amplified target DNA isused as a source for ligation of standard PLPs. The ligated padlockprobes are hybridized on the array and labelled with e.g. astreptavidin-coupled fluorescent probe Alexa (532) directed against thedesthiobiotin moiety of the padlock. This method comprises anexonuclease step after the ligation and a NaOH denaturation step beforecapturing of the padlock nucleotide probes. The elution can be performedwith a 80° C. step in H₂O or with biotin. If the unique cleavablesequence is a poly-uracil sequence, cleavage can preferably be affectedby treatment with uracil-N-glycosidase and endonuclease IV (see FIG. 21and FIG. 22).

The presence or absence of ligated padlock probes can also be visualizedwith BCA (bio-bar-code amplification, Jwa-Min Nam et al., supra). BCA isa PCR-less target amplification method that relies on noveltwo-component oligonucleotide-modified gold nanoparticles (NPs) andsingle-component oligonucleotide-modified magnetic microparticles (MMPs)and subsequent detection of amplified target DNA in the form of bar-codeDNA using a chip-based detection method (see FIG. 23).

In this BCA detection reaction, target DNA is used as a source forligation of standard PLPs. This method comprises an exonuclease stepafter the ligation and a NaOH denaturation step before capturing of thepadlock nucleotide probes. The second member of a binding pair will bindthe first member of a binding pair which is available on the PLP. Ifsaid first member is desthiobiotin, said second member is streptavidin.If the unique cleavable sequence is a poly-uracil sequence, cleavage canpreferably be affected by treatment with uracil-N-glycosidase andendonuclease IV. The linear PLP stays bound to the streptavidin, thegoldbeads bearing both ZIP codes complementary to the nucleotidesequence in the padlock and fluorescent barcode nucleotides, willhybridize (FIG. 24-25) Recently with the use of Nanoparticle-BasedBio-Bar Code technology proof-of-principle has been shown that both DNAand protein can be detected with PCR sensitivity without enzymaticamplification reaction (Christine D. Keating. Nanoscience enablesultrasensitive detection of Alzheimer's biomarker. PNAS, Feb. 15, 2005,2263-2264).

Typical preferred PLPs of the invention are the standard Padlock probe,the PRI-lock probe and the LUNA-probe as depicted in FIG. 7. The probescan be constructed using normal genetic engineering techniques, such asdisclosed in handbooks like Sambrook, J., Fritsch, E. F., and Maniatis,T., in Molecular Cloning: A Laboratory Manual. Cold Spring HarborLaboratory Press, NY, Vol. 1,2,3 (1989). Oligonucleotides which need tobe assembled for use in the present invention can be made syntheticallyby standard DNA or RNA chemical synthesizers or may be obtained fromenzymatic digestion of wild-type, naturally occurring sequences. It isalso possible that naturally occurring sequences are modified byinsertion, substitution or deletion of one or more nucleotides usingconventional genetic engineering techniques.

In the general description of these probes herein below and in theExamples, a poly-uracil sequence is taken as the unique cleavage site,and a desthiobiotin moiety is taken as a first member of a binding pair.It should however be understood that alternatives for these embodiments,as discussed above, are also within the scope of this invention.

Insertion of the poly-uracil sequence as described above can be done bystandard techniques, as mentioned above, which techniques are well knownto a person skilled in the art.

If a desthiobiotin moiety is used, it is preferably obtainedcommercially. These moieties are commercially available as desthiobiotincoupled to a thymidine nucleotide. This nucleotide is introduced intothe padlock nucleotide probe according to standard methods.

Below the working of these probes in the present invention will beexplained in more detail.

The Basic Principle—the Standard Padlock Probe

Standard padlock probes (PLPs) according to the general structure asdepicted in the top figure of FIG. 7 are designed in such a way that aset of those PLPs all comprise the same universal forward and reverseprimer binding sites, designated as universal forward and reverse primerbinding sites. Choice of these primer binding sites is flexible, insofarthat care should be taken that the primer binding site sequences differsubstantially from the rest of the padlock probe, so that theamplification step, which makes use of the universal primers is nothampered by homologous sequences in the rest of the probe.

In each standard PLP a unique set of target specific sequences isinserted, which is designed to bind to a specific target sequence whichis suspected to be present in the sample. The target specific sequenceshould be unique, meaning that it can hybridise with only one targetnucleotide molecule in the sample. Of course, care should be taken thatthe target specific sequences T1 and T2 are directed and designed insuch a way that, upon hybridisation with the target nucleotide in thesample, they are able to be ligated to each other. This in particularmeans that either the T1 sequence should be in the sense direction (i.e.recognising the target in the 5′ to 3′ direction) and the T2 sequence inthe anti-sense direction, or vice-versa. Further, the PLP comprises aunique ZIP-code, which eventually will serve for the detection of thePLP. There is no functional restriction with regard to this ZIP-code,other than that each PLP of the set of PLPs should have a uniqueZIP-code and that it can serve for detection in the assay. For thispurpose, preferably the ZIP-codes used for a given set of PLP-probesshould be of the same size and character, in order not to influence theother steps of the method, such as the amplification step. Preferably,the ZIP codes are derived chosen from the GeneFlex™ TagArray set(Affymetrix) or any other similar library.

The other elements of the PLP (uracil-site, desthio-biotin moiety) areas described above.

Once a set of these probes is produced they can be added to a sampleunder conditions which are optimal for alignment and hybridisation ofthe target specific sequences T1 and T2 to the target sequences in thesample. When the hybridisation reaction is complete the PLPs that havehybridised to a target sequence are ligated by addition of the enzymeligase to the reaction mixture. Thereafter, preferably the non-ligatedDNA is removed by exonuclase degradation. This exonuclease treatment canbe performed with either a 3′ to 5′ exonuclease or a 5′ to 3′exdonuclease or both or an exonuclase which combines both activities. Itis paramount for the present invention that these exonuclease(s) do nothave any endonuclease activity.

Next, the probes are captured using e.g. streptavidin coupled magneticbeads (or another streptavidin coated solid support, such as a column orfilter upon which streptavidin is immobilised) and separated from thesample. Subsequently, the probes are cleaved at the uracil-site byadding a sufficient amount of uracil-N-glycosidase and endo IV nuclease.

This in particular means that ZIP-code probe region of the unligatedpadlock probes is removed, while the ligated probes are linearized. Theprobes are then eluted from the beads by using an aqueous solution ofbiotin or a 80° C. treatment in H₂O.

Finally, the eluted probes are amplified with PCR using the universalprimers (one of which is labelled). Amplicons are then hybridised one.g. micro-arrays on which sequences which are complementary to the ZIPsequences are spotted.

The PRI-Lock Probe

The general structure of the PRI-lock probe is given in the middlesection of FIG. 7, with the remark that the universal Zip-code is anoptional feature, as will be explained below. Note that the differencewith the above standard Padlock probe is that now the primer bindingsites are unique primer binding sites, while the optional ZIP-code isuniversal.

All the other elements of the PRI-lock probe are similar to those of thestandard Padlock-probe and can be applied as mentioned above. Also thehybridisation, ligation, linearization and elution of the PRI-lock probeis identical to those described above.

The Universal ZIP-code is designed for being able to hybridise to auniversal probe, such as a TaqMan probe.

The scheme of the applied procedure is outlined in FIG. 9. MultiplePRI-lock probes are ligated on fragmented target DNA. Target recognitionis achieved by specific hybridization of both arm sequences, andefficient ligation occurs only if the end nucleotides are perfectlymatching to the target. Therefore, the probes confer superiorspecificity. After ligation, the probes are captured onstreptavidin-coated magnetic beads via the desthiobiotin, and arecleaved at the deoxy-uracil nucleotides. The ligation mix and the TaqManprobe region of unligated probes are removed by several washing steps,eliminating the background due to the presence of unligated probes. Theremaining probes are eluted in aqueous biotin solution or after a 80° C.incubation step, the ligated probes are assayed in real-time PCR using aunique primer pair for each target.

The linear quantification range of the proposed procedure is dependenton both the ligation step and the real-time PCR. Ligation ofoligonucleotides has been shown to reflect well the target quantity andwas used successfully for characterization of gene expression and genecopy number in a multiplex setting.

PRI-locks combined with the OpenArray™ system (BioTrove) are useful fora flexible and easily adaptable design of high-throughput, quantitativemultiplex DNA assays, since the target recognition step is separatedfrom downstream processing. The primer binding and TaqMan probe siteswere chosen from a set of artificial, well-balanced sequences that hadbeen selected to have minimum cross-hybridization (e.g. the GeneFlex™TagArrays set (Affymetrix))

Such a design ensures ideal amplification under universal PCR conditionsand enables the use of a single, universal TaqMan probe. Therefore, thesystem is cost-efficient, easily modifiable and extendable to othertargets, even newly emerged pathogens.

It is also possible to omit the universal ZIP-code from the PRI-lockprobe. In that case, detection of a successful amplification is achievedby detecting double-stranded DNA. There are numerous ways to detectdouble-stranded DNA, but in the art this is mostly achieved by usingso-called intercalating dyes, i.e. dyes which only adhere to thenucleotides when these are double-stranded. The most known example ofsuch a dye is ethidiumbromide (EtBr), but use of this dye isdisadvantageous because of its toxicity. A useful alternative is SYBRGreen, a commercially available dye (e.g. from Invitrogen or AppliedBiosystems). Addition of the dye to the PCR mixture gives an easyread-out if double-stranded DNA is present in the mixture, which isindicative of a successful amplification, i.e. presence of the target.

The LUNA probe

The LUNA probe is also a variant of the above described standard Padlockprobe, the difference being that the universal forward primer bindingsite comprises a T7 polymerase recognition site. When the hybridisationreaction is complete the PLPs that have hybridised to a target sequenceare ligated by addition of the enzyme ligase to the reaction mixture.Thereafter, preferably the non-ligated DNA is removed by exonuclasedegradation. This exonuclease treatment can be performed with either a3′ to 5′ exonuclease or a 5′ to 3′ exdonuclease or both or an exonuclasewhich combines both activities. It is paramount for the presentinvention that these exonuclease(s) do not have any endonucleaseactivity. Next, the probes are captured using e.g. streptavidin coupledmagnetic beads (or another streptavidin coated solid support, such as acolumn or filter upon which streptavidin is immobilised) and separatedfrom the sample. Subsequently, the probes are cleaved at the uracil-siteby adding a sufficient amount of uracil-N-glycosidase and endo IVnuclease. By addition of the reverse primer and the presence of AMVreverse transcriptase the T7 site becomes accessible for the RNApolymerase which is common for all the LUNA probes and is used asstarting point for a generic NASBA amplification at a fixed temperature(Compton, J, 1991, Nature 350: 91-92.; Kievits, T, van Gemen, B, VanStrijp, D, Schukkink, R, Dirks, M, Adriaanse, H, Malek, L, Sooknanan, Rand Lens, P. 1991, J. Virol. Methods 35: 273-286; Leone, G, vanSchijndel, H, van Gemen, B, Kramer, FR, and Schoen, C D. 1998, NucleicAcids Research 26: 2150-2155.). A NASBA reaction is based on theconcurrent activity of AMV reverse transcriptase (RT), RNase H and T7RNA polymerase, together with two primers to produce amplification (3).This process occurs at one temperature (41° C.)

The generated ssRNA's are individually detected via the designedidentifier sequences (ZIP-Code), which allows unique target dependentidentification. FIG. 14 depicts a generalised assay method with theseLUNA probes.

Two different approaches (FIG. 19) for detection can be followed toanalyse the amplification products: i) on array or ii) in solution.

i) On Array.

With the array based multiplexing approach the amplified products arecollected on different matrices (array or Luminex beads) (Gordon R F,McDade R L, 1997, Multiplexed quantification of human IgG, IgA, and IgMwith the Flowmetrix system. Clinical Chemistry, 43: 1799-1801) wherecomplementary ZIP (cZIP)-Code oligonucleotides with free 3′ ends havebeen immobilized (FIG. 14.). With arrays probe addressable sites areused for target identification. Luminex beads are color coded beads; theassociation of the amplified product with a characteristic Luminex beadis used as a tag for target identification. With the Luminex approach wehave an array in solution. The introduction of cZIP-Code sequences(arbitrarily non-target sequence of approximately 20-25 nucleotides)makes detection of amplified products on both matrices independent fromtarget sequences. As the array or Luminex beads containing cZIP-Codesare independent, this makes the described assay system adaptable fordifferent fields of application.

ii) In Solution

With the solution based multiplexing approach amplification of reactedpadlock probes is performed by a universal NASBA at a fixed temperature.The ZIP-Code of every different padlock probe is used as a recognitionsite for a molecular beacon in NASBA (Leone, G. et al, supra). Molecularbeacons are single-stranded oligonucleotides having a stem-loopstructure. The loop portion contains the sequence complementary to thetarget nucleic acid, whereas the stem is unrelated to the target and hasa double-stranded structure. One arm of the stem is labeled with afluorescent dye, and the other arm is labeled with a non-fluorescentquencher. In this state the probe does not produce fluorescence becausethe energy is transferred to the quencher and released as heat When themolecular beacon hybridizes to its target it undergoes a conformationalchange that separates the fluorophore and the quencher, and the boundprobe fluoresces brightly (Tyagi, S. and Kramer, F. R. (1996) NatureBiotechnol., 14, 303-308).During this amplification fluorescence based identification isperformed. The combination of PLPs with NASBA is new for multiplexdetection of different RNA/DNA target sequences and has been named LUNA:Ligase dependent Universal NASBA (FIG. 14). Especially the incorporationof the T7 recognition site into the PLP and the use of a universal NASBAgenerating ssRNA is a new feature in the development of an efficientmultiplex detection method, which allows lower target detection levels.Combining this technique with micro-array technology makes it auniversal tool for multiplex analysis for different targets.

EXAMPLES Example 1 Multiplex Diagnosis of Plant PathogenicMicro-Organisms Nucleic Acids Used in the Study

The pathogenic organisms were derived from the culture collection of theapplicant. (Table 1) Genomic DNAs were extracted as previously described(Bonants, P., Hagenaar-de Weerdt, M., van Gent-Pelzer, M., Lacourt, I.,Cooke D. and Duncan, J. (1997) Detection and identification ofPhytophthora fragariae Hickman by the polymerase chain reaction. Eur. J.of Plant Pathol., 103, 345-355.).

Padlock Probe Design

Relevant nucleic acid sequences derived from Genbank and fromindependent sequencing studies were aligned by using ClustalW,implemented in BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).Diagnostic sequences were identified for each target group. PotentialPLP target complementary regions were selected in a way that thediscriminatory nucleotides would bind to the 3′ arm region, and to matchcertain stability criteria (see Results). Melting temperatures (T_(m))to characterize binding strengths of arm sequences were calculated usingthe nearest neighbour method, as implemented in Hyther™(http://ozone2.chem.wayne.edu/). The prediction parameters were set tomatch ligation conditions ([Na]=0.025 M; [Mg²⁺]=0.01 M; T=65° C. and[oligo]=2.5*10⁻¹¹M). Specificity was ensured by positioning a stronglydestabilizing mismatch at the PLP 3′end with closely related, non-targetsequences. PLPs with target oligonucleotides as listed in Tables 2A and3A, were synthesised by Eurogentec S. A. (Seraing, Belgium). The PLP armsequences were combined with the universal primer binding sites (P1: 5′CTCGACCGTTAGCAGCATGA 3′; P2: 5′ CCGAGATGTACCGCTATCGT 3′) and a ZipCodesequence. The unique identifier was chosen from GeneFlex™ TagArray set(Affymetrix) in a way to minimize PLP secondary structures. Secondarystructure predictions were performed by using MFold(http://www.bioinfo.rpi.edu/applications/mfold/). When necessary, PLParm sequences were also adjusted to avoid strong secondary structuresthat might interfere with efficient ligation.

Ligation and Exonuclease Treatment

Genomic DNA was fragmented by digestion using EcoRI, HindIII and BamHI(New England Biolabs) for 30 min, and used as template in the indicatedamount. Cycled ligation was performed in 10 μL reaction mixturecontaining 20 mM Tris-HCl pH 9.0, 25 mM KCH₃COO, 10 mM Mg(CH₃COO)₂, 10mM DTT, 1 mM NAD, 0.1% Triton X-100, 20 ng sonicated salm sperm DNA, 2.4U Taq ligase (New England Biolabs) and 25 pM PLP. For multiplexdetection the concentration of the individual PLPs were adjusted toachieve comparable performance, and ranged from 25 to 200 pM. Reactionsmixtures were made up on ice, and transferred rapidly to a thermalcycler. After 5 min at 95° C., 20 cycles of 30 sec at 95° C. and 5 minat 65° C. were performed, followed by 15 min inactivation at 95° C.After ligation, 10 μL of exonuclease mix (10 mM Tris-HCl pH 9.0, 4.4 mMMgCl₂, 0.1 mg/ml BSA, 0.5 U Exonuclease I (USB) and 0.5 U ExonucleaseIII (USB) was added to each reaction, and the samples were incubated at37° C. for 2 h, followed by inactivation at 95° C. for 2.5 h.

Real-Time PCR

Amplification of ligated PLPs was followed in real-time using an ABIPrism 7700 Sequence Detector System (Applied Biosystems) and the qPCRkit (Eurogentec). Reaction mixtures of 25 μL contained 2.5 μL, 10×real-time buffer, 3 mM MgCl₂, 200 nM of each dNTP including dTTP/dUTP,100 nM P-Frag TaqMan probe (5′ FAM-CCCGGTCAACTTCAAGCTCCTAAGCC-TAMRA 3′),300 nM of primers P1-f20 (5′ CCGAGATGTACCGCTATCGT 3′) and P2-r20 (5′TCATGCTGCTAACGGTCGAG 3′), 0.6 U Hot Gold Start polymerase, 0.6 U UNG and3 μL ligation-exo mix as template. The reaction mix was initiallyincubated at 50° C. for 2 min, followed by 10 min denaturation at 95°C., and 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Fluorescencewas recorded in the second step of each cycle.

Late-PCR

For microarray hybridisation, circularised PLP probes were amplified inLATE-PCR (linear-after-the-exponential PCR) (Sanchez, J. A., Pierce, K.E., Rice, J. E. and Wangh, L. J. (2004) Linear-after-the-exponential(LATE)-PCR: an advanced method of asymmetric PCR and its uses inquantitative real-time analysis. Proc. Natl. Acad. Sci. U.S.A., 101,1933-1938.) to produce a large amount of ssDNA amplicons. Lengths of theprimers were adjusted so that they would have similar meltingtemperatures despite the 10-fold concentration difference. PLPs wereamplified in 25 μL reaction mixtures containing 1× Pfu buffer(Stratagene), 200 nM of each dNTP, 500 nM of Cy3- or Cy5-labeled P1-f19primer (5′ CGAGATGTACCGCTATCGT 3′), 50 nM P2-r20 primer, 0.375 U Pfu(Stratagene) and 3 μL ligation-exo mix as template. The temperatureprofile of the reaction was: 5 min at 95° C., 40 cycles of 2 sec at 51°C., 5 sec at 72° C. and 15 sec at 95° C., after which the reaction wasimmediately cooled to 10° C. PLP amplicons were analysed by agarose gelelectrophoresis before applying them on array.

Microarray Preparation

Complementary ZipCode (cZipCode) oligonucleotides (FIG. 4) carrying aC12 linker and a 5′ NH₂ group were synthesised and spotted on NexterionMPX-E16 epoxy-coated slides by Isogen B. V. (Utrecht, The Netherlands)according to manufacturer's instructions (Schott Nexterion). Briefly, 50nL of 1.5 mM cZipCode solution was spotted using an OmniGrid100contact-dispensing system (Genomic Solutions) equipped with SMP4 pins(Telechem) at 50% relative humidity. After 1 hour incubation at 75%humidity, the uncoupled probes were removed by washing in 300 mM bicine,pH 8.0, 300 mM NaCl, and 0.1% SDS for 30 min at 65° C., followed byrinsing with deionised water and drying by spinning at 250 g for 2 min.The arrays were stored in dark, in a desiccator at room temperatureuntil use.

Microarray Hybridisation

Prior to hybridisation, the arrays were washed and the functional groupswere blocked according to manufacturer's instructions. The hybridisationmixes were made up of 5 μL Cy3-labeled sample and 5 μL Cy5-labeledbackground control sample in 3 M TMAC, 0.1% sarkosyl, 50 mM Tris-HCl pH8.0, 4 mM Na₂EDTA. Cy5-labeled hybridisation control was added to 20 pMfinal concentration in 50 μl final volume. For each slide, one of thehybridisation samples contained Cy5- and Cy3-labeled ampliconscorresponding to the same, positive ligation reaction, which served tocorrect for dye bias (dye correction sample). The mixes were heated for10 min at 99° C. and cooled down rapidly on ice. Sixteen-well siliconsuperstructures (Schott Nexterion) were attached to the arrays to createseparate chambers for the subarrays. After adding 40 μl of the samplesto each well, the chambers were sealed, and the arrays were hybridisedat 55° C. o/n in high humidity. Afterwards, the isolators were removed,and the slides were washed once at 55° C. for 5 min in prewarmed1×SSC/0.2% SDS, and twice for an additional 1 min at RT in 0.1×SSC/0.2%SDS and in 0.1×SSC, respectively. Finally, the slides were dried byspinning at 250 g for 2 min.

Analysis of Microarray Data

Microarrays were analysed using a confocal ScanArray® 4000 laserscanning system (Packard GSI Lumonics) containing a GreNe 543 nm laserfor Cy3 and a HeNe 633 nm laser for Cy5 fluorescence measurement. Laserpower was fixed at 70% for both lasers, while PMT (photomultiplier tubepower) ranged from 45 to 65%, depending on signal intensity. Fluorescentintensities were quantified by using QuantArray® (Packard GSI Lumonics),and the parameters ‘mean signal-mean local background’ (mean Cy3-B ormean Cy5-B) and the ‘mean local background’ (B) were used in furthercalculations. Dye correction factor was calculated separately for eachslide and scanner setting, based on the subarray to which the same butdifferently coloured samples were hybridised (averaged (meanCy3-B)/(mean Cy5-B) based on positive spots). Assay background for theother subarrays was calculated per spot as ‘mean Cy5-B multiplied by thedye correction factor’. Absolute signal intensity was defined as ‘meanCy3-B minus assay background’ and was transformed to log scale(signal=log₂(absolute signal)). If ‘mean Cy3-B’ was lower than ‘assaybackground’ signal was evaluated as zero. To evaluate the significanceof the signal, we compared it to the corresponding assay background, andcalculated log₂(absolute signal/assay background), which was calledreliability factor. The probes were spotted in 3 times triplicates (9parallels). After excluding the outliers, signals and reliabilityfactors were averaged for the probes, and standard deviations (SD) werecalculated. A signal for a probe was called positive if the reliabilityfactor was higher than 1 (i.e. signal was minimum twice the assaybackground) and the mean Cy3 signal was higher than twice the mean localCy3 background (cut-off value).

Results Evaluation of Ligation Specificity and PLP Design Strategies

For diagnostic applications, the high discriminatory power of theligation is of prime importance, since very similar, non-target DNAmolecules can be present potentially in much higher concentration thanthe target DNA. Therefore, we aimed to optimise the reaction conditionsand PLP design for maximum discrimination of single mismatches, whichsubsequently could be extrapolated to diagnostic assay design.

To characterize the discriminatory power of ligation under assayconditions, we quantified the circularised PLPs by real-time TaqMan PCR(FIG. 1). The quantification range of the real-time PCR was linear overa minimum of five orders of magnitude and the amplification efficiency(E) was found to be 0.81 (FIG. 2, insert).

The experimental system to optimise the ligation conditions consisted ofPLP P-frag, which targeted the ITS region of Phytophthora fragariae, andof the corresponding synthetic, target and non-target oligonucleotides(Table 2A). First, we tested various reaction conditions, and found thatcycled ligation consisting of 20 cycles of 5 minutes at 65° C. providedgood discrimination, sufficient yield of ligation product and freedomfrom potential secondary structures (data not shown). The reactionmixture also contained 20 ng sonicated salmon sperm DNA, which served toprovide a large excess of non-target DNA. All the subsequent experimentswere performed under these conditions. Using oligonucleotides D0-D6 astargets, we tested how the discriminatory power of PLP P-frag dependedon the type and the position of the mismatch (Table 2B). Thediscrimination factor was defined as the fold-difference in the yield ofligation product with target and mismatched oligonucleotides, asdetermined by real-time PCR. In agreement with previous results (10),mismatches positioned at the 3′ end of PLP were strongly discriminating,while those at the 5′ end provided much less specificity. The type ofthe mismatch was also found to be important, although to lesser extent.In general, it appeared that the nearest neighbour parameters could beindicative of the destabilizing effects of different mismatches.Mismatched nucleotide pairs including cytosines were betterdiscriminated, while the G-T pair (at the 5′ end) hardly affected theligation efficiency.

Next, we examined whether different PLP design strategies could furtherimprove the discriminatory power. Apart from the above-describedsymmetric design, we tested two asymmetric design principles. Since weused the same PLP, the effect of variable arm lengths was mimicked bychanging the probe-complementary sequence of the target oligonucleotides(Table 2A). First, we shortened the probe-complementary sequence to the3′ arm to increase its discriminatory power, with a correspondinglengthening of the 5′ arm-complementary sequence to ensure the stablebinding of the probe (oligonucleotides A1 and A1C). The second strategyinvolved inserting a destabilizing mismatch in the middle of the 3′arm-complementary sequence, and the binding of the probe was similarlystabilized by lengthening the 5′ arm (oligonucleotides A2 and A2C). As aconsequence, the melting temperatures (T_(m)) of the 5′ arm sequencesbecame higher than the reaction temperature, while those of the 3′ armswere about 20 to 30° C. below it (Table 2C). We believe that these T_(m)values are only indicative of the real binding conditions, since thehybridisation of the 5′ arm of PLP makes the binding of the 3′ armalmost a unimolecular reaction. We hypothesized that such designprinciples would result in an equilibrium process between a bound and anunbound 3′ arm, which could increase specificity. As expected, bothasymmetric design strategies significantly increased the discriminatorypower of PLP P-frag against non-target oligonucleotides with 3′ C-Cmismatches (Table 2C and FIG. 2). Shortening the 3′ arm sequence provedto be more efficient, since it did not reduce the ligation yield as muchas the internal mismatch. Using this strategy we could achieve adiscrimination factor of 1477 as compared to 175, provided by thesymmetric design. Therefore, we selected this PLP design to generateprobes for our diagnostic system to detect plant pathogenic organisms.It is interesting to note that although the internal mismatch resultedin less decrease in T_(m) than the strategy involving a short 3′ armsequence, it reduced the ligation efficiency more. We believe thisphenomenon is due to the perturbation of dsDNA secondary structure,which could hinder the ligation reaction.

Design and Testing of Diagnostic Padlock Probes

Based on the principles described above, we designed PLPs targeting ten,economically important plant pathogens (Table 3A). In each case, weselected discriminatory areas within the ITS regions of rRNA operonsbecause of their high copy number (1), which could significantlyincrease the sensitivity of the assay. Further, ITS regions have beenextensively used in phylogenetic studies (11), and a large number ofsequences are available for plant pathogenic organisms, which may ensurereliable assay design. Sequences available in Genbank and those obtainedfrom independent sequencing studies were aligned, and diagnostic regionsfor each target organism were selected. Preferably, we chose regionscontaining more than one discriminatory nucleotides, and very fewpolymorphic positions within the targeted species/subgroups. The 3′ armsequences were selected to be 14-18 nucleotide-long and had a T_(m)around 40° C. (Table 3B). In general, the 3′ arm sequence hybridised tothe discriminatory region and contained a highly destabilizing mismatchor a gap at the 3′ end when bound to the non-target sequence. The 5′ armsequences were 27-37 nucleotide-long. As an attempt at hierarchicaldiagnostic analysis, we also designed a genus-specific PLP to target allPhytophthora species and discriminate them from related oomycetes. Afterselecting the target-complementary regions, they were combined with theuniversal primer binding site sequences, and a unique ZipCode sequencewas selected for each probe.

The developed probes were tested for sensitivity and discriminatorypower using synthetic oligonucleotides representing target nucleic acidsand the most similar, non-target sequences. Our rationale to test PLPswith oligonucleotides was that it was easy to implement and providedreliable data to compare PLP properties. Further, identification ofcertain subtypes often requires extensive characterization, while otherisolates, mostly those of the closely related non-target organism, mightbe exotic and difficult to obtain. Therefore, we propose that an initialtesting of PLPs with target and non-target oligonucleotides could beadapted as a standard approach.

Since the final analysis was to be performed on array, we chose theLATE-PCR protocol (Sanchez, J. A. et al. supra) to achieve efficientamplification and produce large amount of ssDNA in one step, which isideal for microarray hybridisation. In all the subsequent experimentsthis method was used to amplify ligated PLPs.

Fixed amounts of PLPs were ligated on their respective target and therelated, but non-target oligonucleotides, present in a wideconcentration range (FIG. 3A). Sensitivity of PLP detection was definedas the lowest amount of target oligonucleotide that resulted in apositive signal as assessed by gel electrophoresis. The magnitudedifference between the lowest amount of the target and that of thenon-target oligonucleotide that gave positive signal was called‘discriminatory range’. These experimental characteristics along withthe design parameters for each PLP are shown in Table 3B.

In order to extrapolate these values to the expected specificity inreal-world assays, we also tested PLP sensitivity and discriminatoryrange with a dilutions series of genomic DNA. Since the isolate ofPythium splendens (Genbank accession no. AF310336) that is the mostsimilar non-target organism for PLP Phyt-spp was not available, weperformed this experiment with PLP P-cac, which discriminates P.cactorum and P. nicotianae species based on two nucleotides. Both PLPP-cac and PLP P-nic could successfully detect their targets using 5 pgof genomic DNA, which corresponds to ˜100 genome equivalents (Kamoun, S.(2003) Molecular genetics of pathogenic oomycetes. Eukaryot. Cell, 2,191-199) (FIG. 3B). This experiment proved that the probes have similarand sufficient sensitivities, and the genomic DNAs were of good quality.However, ligation of PLP P-cac even on very high amounts of P.nicotianae genomic DNA (250 ng) did not give rise to any discernible PLPamplicons, indicating strict specificity (FIG. 3C). Since thediscriminatory range of PLP P-cac was among the lowest of those of thedesigned PLP set, we concluded that all the probes must be specific totheir cognate genomic DNA target.

Validation—PLP-Based Multiplex Detection of Plant Pathogenic Organisms

A mix of the developed 11 PLPs was ligated on various genomic DNAs,treated with exonucleases, and subjected to LATE-PCR using Cy3-labeledforward primer. The labelled PLP amplicons were analysed onmulti-chamber, low-density universal microarrays, which enabled thesimultaneous assay of 16 samples on a single slide (FIG. 4). The tagarray used in our experiments contained 30 probes in 9 replicates,together with 90 hybridisation control probes distributed over thedeposition area. This layout allows for the future extension of the PLPset to target other pathogens and enables high-throughput screening (seeFIG. 8).

Because of the great sensitivity of microarray detection, we found thatsignificant fluorescent signal could be detected even when target DNAwas absent from the ligation reaction. Since our results indicated thatthe ligation reaction is highly specific, we concluded that the observedsignal must have been derived from amplification of unligated PLPs thathad not been completely removed by exonuclease treatment (‘backgroundamplification’). This ‘assay background signal’ was comparable to thatmeasured in the absence of ligase, suggesting a ligation-independentmechanism (data not shown). To correct for the ligation-independentsignal and to define the detection threshold of the assay, weincorporated a background control sample, which contained no target DNAin the ligation and was subjected to the same treatment. It was labelledwith Cy5 and hybridised to each array together with the Cy3-labeled PLPamplicons. The assay background signal, measured in the Cy5 channel andcorrected for dye bias, was deducted from the Cy3 signal for each spot.Further, we calculated a reliability factor characterizing the ratio ofsignal and assay background (log₂ (absolute signal/assay background)).

Using the developed PLP set, we tested genomic DNAs from a panel ofwell-characterized isolates of plant pathogenic organisms (Tables 1 and4; FIG. 5 a-g). In each case, 1 ng genomic DNA could be specifically andreliably detected without any cross-reaction. All the Phytophthoraspecies were correctly recognized by PLP Phyt-spp, including P.cactorum, which contained two adjacent mismatches with the 5′ armsequence of the probe (Table 3A). This polymorphism was apparently welltolerated, resulting in a positive signal. For four probes (PLPs P-cac,P-nic, P-inf and V-dahl) analysis was also performed with DNA of a veryclosely related organism (Table 3B), but no cross-reaction was observed,indicating excellent specificity. When there was no cognate target DNApresent to any of the PLPs, we observed a certain level of Cy3 signalfor some of the probes. They were, however, well below the threshold,correctly identifying the samples as negative. In the presence ofligation target, PLPs were circularised and could serve as template inPCR. Consequently, amplification of the ligated PLPs proceededefficiently, suppressing the ligation-independent amplification, and thecorresponding signal for the non-cognate probes was reduced. Thus, usingthe above-described correction method, zero signal was scored for mostprobes when there was ligation target present, due to subtraction ofassay background signal.

Next, we evaluated the ability of the developed diagnostic system todetect several pathogens in parallel. Mixtures of equal amounts ofgenomic DNAs representing three targeted organisms were tested (FIG. 5h-j). In two out of three cases, the pathogens were correctly andunambiguously identified by all four cognate probes, resulting indetection at the genus and species/subgroup level. The components of thethird mixture, P. cactorum, R. solani AG 4-1 and V. dahliae, were alsocorrectly identified by using the species/subgroup-specific probes. ThePLP Phyt-spp signal, however, was below the threshold, most probably dueto the two adjacent mismatches with P. cactorum DNA.

To explore the sensitivity of the system in a multiplexed setting, wetested the detection threshold for F. oxysporum and M. roridum in thepresence of a large excess of the other target DNA (FIG. 5 k-l). Aslittle as 0.5 pg of F. oxysporum DNA could be detected in the presenceof 500 pg M. roridum DNA, corresponding to a dynamic range of 1000. In areverse situation, the detection threshold was 5 pg for M. roridum inthe presence of 500 pg Fusarium DNA, indicating that a reciprocaldynamic range of 100 is achievable using this system.

Example 2 Pilot-Scale Study to Demonstrate Proof-of-Principle PRI-LockProbes Design

Three PRI-lock probes were designed to target economically importantplant pathogens so as to create a pilot-scale, multiplex detectionsystem to test the proposed principle (FIG. 7). A universal TaqManprobe, containing LNA (locked nucleic acids) was designed to monitor theamplification. The specificity of the assay will be demonstrated bytesting DNAs of the most similar, non-target organisms for each probe(Table 5). Since target discrimination is achieved based on only asingle or a few nucleotides, this pilot system also shows the potentialof PRI-Locks for extremely specific, quantitative analysis on auniversal platform.

Specificity of PRI-Locks in Singleplex Reactions

First, we tested the specific ligation of the designed PRI-lock probeson their cognate targets, and the specific amplification by using theunique primer pairs in combination with the universal TaqMan probe. EachPRI-lock probe was ligated on 500 pg target genomic DNA and specificamplifications were observed with Ct values of 22-25, depending on thePRI-lock probe used (FIG. 12). No signal was observed in lack of targetDNA.

Performance of PRI-Lock-Based Detection in a Multiplex Setting

To evaluate the performance of PRI-lock based detection, we carried outmultiplex reactions with single or multiple target pathogens (Table 6).The Ct values observed in real-time PCR were compared to those obtainedin singleplex reactions.

All the pathogens present were specifically detected without exception.The Ct values apparently were not affected by the presence of multipleprobes. Further, multiple target DNAs could also be detected withoutsubstantial change in the observed Ct values, demonstrating the lack ofinhibition due to possible competition in the assay. Practically, itmeans that the dynamic range of detection (the highest ratio of targetsthat is still detectable in a multiplex reaction) is not an issue usingthat system. We also found that the primers were perfectly specific totheir respective ligated PRI-lock probe template, as expected.

Preliminary Experiments to Characterize the Linear Range ofQuantification.

The linear range of quantification was analyzed for all the threePRI-lock probes using dilution series of target DNA. The resultingcalibration curves can be used for quantification of target insubsequent experiments. Currently, we observed that for two out of thethree PRI-lock probes, the linear range of quantification is only 4magnitudes, because at higher target concentrations the ligation yielddoes not increase any more in a linear fashion with the increasingtarget concentration (FIG. 13). In the planned future application, asubstantial increase in the applied PRI-lock probe concentration (100×)is expected to significantly increase the quantification range.

Example 3 Pilot-Scale Study to Demonstrate Proof-of-Principle LunaProbes Background

After the ligation of the LUNA probe on target DNA, any remainingnon-circularized (unligated) padlock probes can be removed byexonuclease treatment followed by capturing of the probes through bybinding of the desthiobiotin moiety with streptavidin magnobeads. Aftera washing step the remaining circularized probes are digested withUracil-N-glycosidase/endo IV nuclease at the position of theincorporated uracil nucleotides between the 5′ T7 RNA polymeraserecognition site and the 3′ end of the universal reverse primer bindingsite. The release of the complementary T7 site, common for allpadlock-probes, acts as starting point for a generic NASBA amplificationat a fixed temperature (see FIG. 14-15)

Design

According to procedures as described above (see Example 1), LUNA probesas shown at the bottom of FIG. 7 were designed.As depicted in FIG. 14, LUNA probe hybridization, ligation, exonucleaseand glycosidase treatment were performed as described previously.Then circularized and linearized LUNA probes are amplified by standardNASBA utilising the T7 primer binding site included in de LUNA probe.Amplified ss products can be hybridized to an array or Luminex beads onwhich cZipCode sequences are spotted/bound. Luminex beads can beanalyzed with flow cytometry.Amplification of ligated LUNA probes in this example is measured usingMolecular Beacons.

Specificity

Based on sequences of Phytophthora cactorum and Verticillium dahliae twopoint mutation specific LUNA probes have been designed. A mutation onthe 3′end of the probe is more discriminatory than when a mutation isplaced at the 5′ end. Specificity of the probe is largely increased withan asymmetrical design and a high ligation temperature. In an optimaldesign the 3′ arm of the LUNA probe has a melting temperature (Tm) of37-40° C., the 5′-arm has a Tm of 65-70° C. The specificity of the LUNAligation step has been validated with closely related (non)pathogenicspecies and appeared to point mutation specific.The secondary structure and in particular the localization of the T7polymerase recognition site of the LUNA probe is essential for anefficient initiation of the NASBA amplification. Linearization of theLUNA probe by Uracil-N-glycosidase/endo IV nuclease followed byselective capturing of the probes with Streptavidine coated magnobeadsappeared to be essential for an efficient NASBA amplification reaction.The target specific Zip-Codes of the LUNA probes have been used ashybridization sites for the used Molecular Beacons. With thoseidentification tags quantification of the isothermal NASBA could befollowed. As identifiers of the two LUNA probes against P. cactorum andV. dahliae a FAM- and JOE-labeled MB respectively have been designed(FIG. 16).

Performance

The multiplexibility and dynamic detection range are importantparameters of this LUNA detection system. To validate those parametersgenomic DNA of the plant pathogenic Phytophthora cactorum andVerticillium dahliae have been extracted and tested in differentconcentrations and ratio's and compared with traditional PCR (FIG. 17,18).Multiplexing of both targets appeared to be possible; the detectionlimit for both targets appeared to be in the pg range (FIG. 17). Thedynamic detection range for those pathogens was at least 100 (FIG. 18).For target identification the next step will be hybridization of thessRNA LUNA amplicons to arrays spotted with cZipCode oligos or todifferent Luminex beads coupled with different cZipCode oligos.Detection can then be performed by array scanning, flow-cytometry orMolecular Beacon detection (FIG. 19).

Tables

TABLE 1 Isolates of plant pathogenic species and subgroups used inExample 1. Phylum Order Species Isolate Oomycota PeronosporalesPhytophthora nicotianae PRI 28.8 Phytophthora cactorum PRI 18.1Phytophthora infestans VK98014 Phytophthora sojae F. Govers 6497 Pythiumultimum N2001/5 Basid- Ceratobasidiales Rhizoctonia solani AG 4-1 PRI4R91 iomycota Rhizoctonia solani AG 4-2 PRI 4R22 Ascomycota HypocrealesFusarium oxysporum f. sp. .364N2 radicis-lycopersici Myrothecium roridwnPRI 15.2 Phyllacorales Verticillium dahliae 809.97 Verticilliumalboatrum Vet98/ resp.VD5 Nematoda Tylenchida Meloidogyne hapla HBA

TABLE 2Evaluation of single mismatch discrimination by PLP P-frag and optimization ofPLP design strategy. (A)

(B) Target 5′arm 3′arm Discrimination oligo L(nt) T_(m) L(nt) T_(m) CtΔCt (Ct_(x)-Ct_(D0)) factor D0 20 59.2 19 53.1 19.6 ± 0.8 na na D1 2059.2 18 50.9 27.6 ± 0.5 8.0 115 D2 20 59.2 18 50.4 28.3 ± 0.4 8.7 175 D320 59.2 18 50.9 26.6 ± 0.1 7.0 64 D4 19 58.4 19 53.1 21.7 ± 0.6 2.1 4.0D5 19 57.4 19 53.1 19.9 ± 0.6 0.3 1.2 D6 19 57.9 19 53.1 26.2 ± 0.3 6.650 (C) Target 5′arm 3′arm ΔCt (Ct_(x)- Discrimination Design oligo L(nt)T_(m) L(nt) T_(m) Ct Ct₀) factor Symmetric D0 20 59.2 19 53.1 19.6 ± 0.8na na D2 20 59.2 18 50.4 28.3 ± 0.4  8.7 175 Asymmetric I. A1 25 67.0 1539.6 19.6 ± 0.2 na na A1C 25 67.0 14 35.2 31.9 ± 0.4 12.3 1477 Asymmetric II. A2 25 67.0 18 46.8 20.8 ± 0.7 na na A2C 25 67.0 17 43.632.2 ± 0.3 11.4 866 (A) PLP P-frag and target oligonucleotides used tocharacterize ligation specificity. PLP sequence is drawn to show targetcomplementary regions; 5′ and 3′ ends are indicated. Mismatches in thecomplementary oligonucleotide sequences are emphasized by invertedcolours. Lines and slashes indicate continuous sequences. (B) Effect ofposition and type of mismatch on ligation efficiency and mismatchdiscrimination. Lengths (L) and melting temperatures (T_(m)) of PLPtarget-complementary regions are indicated. The Ct values to quantifyligated PLPs were determined in three independent experiments. The meansof Ct values are shown along with the standard deviations to indicatereproducibility. Discrimination factor was calculated as fold-differencein the yield of ligation product, determined based on ΔCt values andamplification efficiency (E = 0.81). (C) Comparison of PLP designstrategies. PLP characteristics are shown as at (B). In the calculationof ΔCt, Ct₀ refers to Ct of ligation reaction with the respectiveperfectly matching target.

TABLE 3 (A) Targeted species/ 3′ target complementary group 5′ targetcomplementary sequence (5′-3′) sequence (5′-3′) ZipCode sequence (5′-3′)

(B) 5′arm 3′arm Length Length # of Sensitivity Discriminatory Name (nt)T_(m) (° C.) (nt) T_(m) (° C.) Closest non-target relative discr.nt (fM)range PLP P-spp 29 60.0 14 39.5 Pythium splendens  1 2   10⁵ PLP P-cac37 65.8 18 39.6 Phytophthora nicotianae  2 20   10⁵ PLP V-dahl 28 58.314 35.4 Verticillium alboatrum  2 2   10⁵ PLP P-inf 28 68.9 17 44.0Phytophthora sojae  3 2   10⁵ PLP F-oxy 27 68.9 15 41.5 Fusariumequiseti  3 2   10⁵ PLP Myr-ror 27 69.0 15 46.5 Myrothecium verrucaria 5 0.2 10⁷ PLP P-nic 36 61.0 14 43.6 Phytophthora cactorum  7 2   10⁶PLP Rhiz-4-2 36 59.2 15 41.0 Rhizoctonia solani AG 4-1  7 2   nt PLPRhiz-4-1 32 66.2 15 39.5 Rhizoctonia solani AG 4-2  8 2   nt PLP Pyt-u32 66.2 15 40.6 Pythium splendens 10 0.2 nt PLP Mel-h 30 65.7 17 41.1Meloidogyne incognita 15 2   nt (A) Target-complementary regions andZipCode sequences of the developed diagnostic PLPs. Nucleotides or gapsdue to deletions used to discriminate targets from most similar,non-target sequences are underlined. Gray boxes indicate polymorphismwithin the target group. (B) Design and experimental characteristics ofthe PLP set. Probes were named after the targeted species/subgroup.Lengths (L) and melting temperatures (T_(m)) of PLP target-complementaryregions are indicated. The number of nucleotides discriminating thetargeted sequence from that of the known most similar, non-targetorganism is shown for each PLP. Sensitivity was defined as the lowestconcentration of perfectly matching oligonucleotide that could bedetected under standard assay conditions. Discriminatory range gives themagnitude difference between the lowest detectable concentrations oftarget and of non-target oligonucleotides.

TABLE 4 Pathogen detection using the developed multiplexed PLP set asanalyzed on microarrays. For each sample and probe combination the meansignal (±SD) of 9 replicates is shown in the upper row left, wheresignal was calculated as log₂ (mean Cy3 − local background − assaybackground). Reliability of the method was evaluated by calculating log₂(absolute signal/assay background) for each spot, which is calledreliability factor. Means (±SD) are shown in the lower row, aligned toright. Criteria for positivity were: mean Cy3 > 2* mean Cy3 localbackground (cut-off value) and reliability factor >1. Positive signalsare highlighted and shown in bold. (A) Analysis of DNAs of singlepathogens. (B) Testing artificial mixtures of pathogen DNAs present inequal ratios. (C) Dynamic range of detection: analysis of DNAs ofMyrothecium roridum and Fusarium oxysporum in different ratios. Signalscorresponding to PLP probes Samples P-spp P-nic P-cac P-inf Pyt-uRhiz-4-1 (A) P. nicotianae, 14.7 ± 0.2 13.8 ± 0.2 0 0 0 0 1 ng 3.9 ± 0.13.5 ± 0.1 na na na P. cactorum, 1 ng 12.1 ± 0.3 0 12.8 ± 0.2 0 0 0 1.4 ±0.2 na 3.5 ± 0.3 na na P. infestans, 1 ng 14.8 ± 0.3 0 0 12.8 ± 0.2 0 05.2 ± 0.1 na na 5.9 ± 0.2 na P. sojae, 1 ng 13.9 ± 0.4 0 0 0 0 0 8.3 ±0.2 na na na na Pyt. ultimum, 0 0 0 0 12.8 ± 0.6 0 1 ng na na na na 6.1± 0.1 R. solani AG 4-1, 0 0 0 0 0 14.3 ± 0.2 1 ng na na na na na R.solani AG 4-2 0 0 0 0 0 0 1 ng na na na na na V. dahliae, 1 ng 0 0 0 0 00 na na na na na F. oxysporum 0 0 0 0 0 0 1 ng na na na na na M.roridum, 1 ng 0 0 0 0 0 0 na na na na na M. hapla, 1 ng 0 0 0 0 0 0 nana na na na V. alboatrum, 10.1 ± 0.5 8.7 ± 0.6 7.3 ± 0.9 7.0 ± 1.0 0 0 1ng −0.1 ± 0.1 −3.0 ± 0.6 0.3 ± 0.2 −1.4 ± 0.5 na No target 10.4 ± 0.49.6 ± 0.4 0 4.7 ± 1.4 0 0 (neg. control) −0.1 ± 0.2 −0.9 ± 0.2 na −2.7 ±1.5 na (B) P. infestans, 11.6 ± 0.4 0 0 11.0 ± 0.4 0 0 500 pg R. solani4-2, 500 pg M. roridum, 2.2 ± 0.1 na na 4.6 ± 0.1 na 500 pg P.nicotiane, 13.0 ± 0.4 12.3 ± 0.4 0 0 11.9 ± 0.4 0 500 pg Pyt. ultimum,500 pg M. hapla, 500 pg 3.1 ± 0.1 2.4 ± 0.2 na na 6.5 ± 0.6 P. cactorum,8.5 ± 1.2 0 11.8 ± 0.2 0 0 13.5 ± 0.4 500 pg R. solani 4-1, 500 pg V.dahliae, −2.6 ± 1.4 na 6.3 ± 0.2 na na 500 pg (C) M. roridum, 0 0 0 0 00 500 pg F. oxysporum, na na na na na 500 pg M. roridum, 0 0 0 0 0 0 500pg F. oxysporum, na na na na na 50 pg M. roridum, 0 0 0 0 0 0 500 pg F.oxysporum, na na na na na 5 pg M. roridum, 0 0 0 0 0 0 500 pg F.oxysporum, na na na na na 0.5 pg M. roridum, 5 pg 0 0 0 0 0 0 F.oxysporum, na na na na na 500 pg M. roridum, 0 0 0 0 0 0 0.5 pg F.oxysporum, na na na na na 500 pg Signals corresponding to PLP probesSamples Rhiz-4-1 Rhiz-4-2 V-dahl F-oxy Myr-ror Mel-h (A) P. nicotianae,0 0 0 0 0 1 ng na na na na na na P. cactorum, 1 ng 0 0 0 0 0 na na na nana na P. infestans, 1 ng 0 0 0 0 0 na na na na na na P. sojae, 1 ng 0 00 0 0 na na na na na na Pyt. ultimum, 0 0 0 0 0 1 ng na na na na na naR. solani AG 4-1, 0 0 0 0 0 1 ng 3.8 ± 0.1 na na na na na R. solani AG4-2 14.5 ± 0.2 0 0 0 0 1 ng na 8.3 ± 0.1 na na na na V. dahliae, 1 ng 012.9 ± 0.7 8.0 ± 0.4 0 0 na na 4.6 ± 0.2 0.7 ± 0.7 na na F. oxysporum 00 15.2 ± 0.2 0 0 1 ng na na na 10.8 ± 0.1 na na M. roridum, 1 ng 0 0 014.3 ± 0.4 0 na na na na 9.3 ± 0.2 na M. hapla, 1 ng 0 0 0 0 15.0 ± 0.2na na na na na 6.5 ± 0.1 V. alboatrum, 8.7 ± 0.5 9.0 ± 1.0 6.0 ± 0.4 7.1± 0.7 7.5 ± 0.4 1 ng na −1.1 ± 0.4 −0.6 ± 0.2 −1.3 ± 0.9 0.1 ± 0.2 −0.5± 0.5 No target 10.6 ± 0.5 8.0 ± 0.5 0 6.8 ± 0.7 9.1 ± 0.8 (neg.control) na 0.1 ± 0.2 −0.9 ± 0.3 na −1.7 ± 0.3 −0.1 ± 0.3 (B) P.infestans, 12.2 ± 0.6 0 0 12.8 ± 0.5 0 500 pg R. solani 4-2, 500 pg M.roridum, na 5.1 ± 0.1 na na 6.3 ± 0.2 na 500 pg P. nicotiane, 0 0 0 012.0 ± 0.4 500 pg Pyt. ultimum, 500 pg M. hapla, 500 pg na na na na na6.8 ± 0.3 P. cactorum, 0 12.4 ± 0.2 0 0 0 500 pg R. solani 4-1, 500 pgV. dahliae, 3.3 ± 0.1 na 5.1 ± 0.1 na na na 500 pg (C) M. roridum, 0 011.2 ± 0.7 14.1 ± 0.7 0 500 pg F. oxysporum, na na na 9.0 ± 1.1 7.0 ±0.3 na 500 pg M. roridum, 0 0 10.6 ± 0.4 13.8 ± 0.7 0 500 pg F.oxysporum, na na na 8.0 ± 0.7 7.5 ± 0.3 na 50 pg M. roridum, 0 0 8.7 ±0.3 12.7 ± 0.4 0 500 pg F. oxysporum, na na na 5.1 ± 0.4 7.8 ± 0.2 na 5pg M. roridum, 0 0 9.0 ± 0.3 13.9 ± 0.6 0 500 pg F. oxysporum, na na na1.5 ± 0.2 8.0 ± 0.3 na 0.5 pg M. roridum, 5 pg 0 0 14.5 ± 0.7 9.6 ± 0.30 F. oxysporum, na na na 10.3 ± 0.2 1.2 ± 0.1 na 500 pg M. roridum, 0 014.5 ± 1.0 5.9 ± 0.9 0 0.5 pg F. oxysporum, na na na 10.0 ± 0.4 −1.5 ±0.5 na 500 pg

TABLE 5 The targeted groups of pathogens and the most similar, non-target micro-organisms for each designed PRI-lock probe. Dis- Closestnon-target crimina- PRI-Lock Target relative tion (nt) PRI_PhytPhytophthora spp. Pythium splendens 1 PRI_P.inf Phytophthora infestansPhytophthora sojae 3 PRI_M.ror Myrothecium roridum Myrotheciumverrucaria 5 The number of discriminating nucleotides are indicated.

TABLE 6 Ct values of real-time PCR reactions performed on ligationmixtures containing different PRI-lock probe and target combinations.Primers Primers Primers Phyt_fw, P.inf_fw, M.ror_fw, Template PRI-LocksPhyt_rv P.inf_rv M.ror_rv Phytophthora PRI_Phyt 24.07 40.00 40.00infestans Phytophthora PRI_P.inf 40.00 22.49 40.00 infestans MyrotheciumPRI_M.ror 40.00 40.00 22.04 roridum Phytophthora PRI_Phyt 24.46 21.9740.00 infestans PRI_P.inf PRI_M.ror Myrothecium PRI_Phyt 40.00 40.0021.59 roridum PRI_P.inf PRI_M.ror Phytophthora PRI_Phyt 25.10 22.7721.46 infestans PRI_P.inf Myrothecium PRI_M.ror roridum No targetPRI_Phyt 40.00 40.00 40.00 PRI_P.inf PRI_M.ror

1.-26. (canceled)
 27. A standard padlock nucleotide probe comprisingfrom 5′ to 3′: a) a target specific nucleotide sequence I (T1) b) ageneric reverse primer binding site c) a nucleotide sequence actingbearing desthio-biotine d) a unique cleavable sequence e) a genericforward primer binding site f) a ZIP-code sequence g) a target specificnucleotide sequence 2 (T2), wherein the T1 and T2 sequences are designedto be complementary to adjacent nucleotide stretches on the same targetin such a way that after hybridization and ligation of the padlock probeforms a circular molecule.
 28. A padlock probe according to claim 27,wherein the unique cleavable sequence is a poly-uracil sequence.
 29. Apadlock probe according to claim 28, wherein the poly-uracil sequencefunctions as a first member of a binding pair.
 30. A padlock probeaccording to claim 27, further comprising a T7 RNA polymeraserecognition site.
 31. A padlock probe according to claim 30, wherein theT7 RNA polymerase recognition site is located 5′ of the generic forwardprimer binding site.
 32. A padlock probe according to claim 27, whereinthe ZIP code is complementary to a nucleotide sequence on an array orother element.
 33. A padlock nucleotide probe (PRI lock) comprising from5′ to 3′ a) a target specific nucleotide sequence I (T1) b) a uniquereverse primer binding site c) a nucleotide sequence bearing adesthio-biotine d) a unique cleavable sequence e) a unique forwardprimer binding site f) a target specific nucleotide sequence 2 (T2),wherein the T1 and T2 sequences are designed to be complementary toadjacent nucleotide stretches on the same target in such a way thatafter hybridization and ligation of the padlock probe forms a circularmolecule.
 34. A padlock probe according to claim 33, wherein the uniquecleavable sequence is a poly-uracil sequence.
 35. A padlock probeaccording to claim 33, which comprises a universal ZIP code situatedbetween the unique forward primer binding site and the target specificnucleotide sequence 2 (T2).
 36. A padlock probe according to claim 27,wherein the poly-uracil sequence comprises from 2-100 nucleotides.
 37. Apadlock probe according to claim 27, wherein the first target specificsequence (T1) has a length of about 10 to about 75 nucleotides.
 38. Apadlock probe according to claim 27, wherein the second target specificsequence (T2) has a length of about 10 to about 30 nucleotides.
 39. Amethod for the detection of a target nucleotide sequence comprising: a.adding to a sample which contains said target one or more padlocknucleotide probes according to claim 1 which have target specificsequences capable of hybridization to said target; b. allowing annealingof the padlock nucleotide probe to said target; c. ligating the padlocknucleotide probe to itself; d. capturing the padlock nucleotide probesvia the desthio-biotin by bringing them into contact with a solidsupport coated with streptavidine e. linearizing the padlock nucleotideprobe; f. washing of the solid support to remove any unboundoligonucleotides g. elution of the PLP probe from the solid support h.detection of the ZIP-code.
 40. Method according to claim 39, wherein thedetection of the ZIP-code comprises the steps of: i. amplification ofthe padlock nucleotide probe using generic primers; j. labelling theamplified padlock nucleotide probe; k. testing for presence of theZIP-code by hybridizing said padlock nucleotide probe with at least onesequence which is capable of hybridization with said ZIP-code, whereinsaid hybridization preferably takes place at a solid support.
 41. Methodaccording to claim 39, wherein detection of the ZIP-code comprises thesteps of: i. testing for presence of the ZIP-code by hybridizing saidpadlock nucleotide probe with at least one sequence which is capable ofhybridization with said ZIP-code, wherein said hybridization preferablytakes place at a solid support; and j. labelling of the hybridizedpadlock nucleotide probe on the solid support with a fluorescent probedirected against the first member of a binding pair of the padlock probeor with fluorescently labelled Biobarcode labelled gold beads.
 42. Amethod for the detection of a target nucleotide sequence comprising: a.adding to a sample which contains said target one or more padlocknucleotide probes which have target specific sequences capable ofhybridization to said target; b. allowing annealing of the padlocknucleotide probe to said target; c. circularization of padlock probe byligation; d. capturing the padlock nucleotide probe via thedesthio-biotine using a solid support coated with streptavidine; e.linearizing the padlock nucleotide probe; f. eluting the padlocknucleotide probe from the solid support, g. amplifying the elutedpadlock nucleotide probe using unique primers; h. monitoring and/ordetection of amplification.
 43. Method according to claim 42, whereinmonitoring and/or detection of amplification is performed using anintercalating dye.
 44. Method according to claim 43, wherein monitoringand/or detection of amplification is performed using a universal probe.45. Method according to claim 39, wherein the unique cleavage sequenceis a polyuracil sequence and wherein linearization is achieved bytreatment with uracil N-glycosidase and endonuclease IV.
 46. Methodaccording to claim 39, wherein biotin is used to elute the probe fromthe solid support.
 47. Method according to claim 39, wherein adenaturation step with NaOH is performed before capturing the padlocknucleotide probes.
 48. Method according to claim 39, wherein the solidsupport is a bead or a column.
 49. Kit comprising multiple padlockprobes according to claim 27, wherein each padlock probe is designed torecognize a unique target.